Cardiac muscle repair or regeneration using bone marrow-derived stem cells

ABSTRACT

Disclosed are compositions and methods for repairing and/or regenerating cardiac tissue by administering adult bone marrow-derived stem cells to an individual. These cells can be administered as a liquid injectible or as a preparation of cells in a matrix which is or becomes solid or semi-solid. The cells can be genetically modified to enhance myocardial differentiation and integration. Also disclosed is a method for replacing cells ex vivo in a heart valve for implantation.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/725,867,filed on Mar. 17, 2010, which is a continuation of application Ser. No.12/395,949, filed on Mar. 2, 2009 (now U.S. Pat. No. 7,892,829, issuedon Feb. 22, 2011), which is a continuation of application Ser. No.10/690,435, filed Oct. 21, 2003 (now U.S. Pat. No. 7,514,074, issued onApr. 7, 2009), which is a continuation-in-part of application Ser. No.10/278,148, filed Oct. 22, 2002 (now abandoned), which is acontinuation-in-part of application Ser. No. 10/127,737, filed Apr. 22,2002 (now abandoned), which is a continuation of application Ser. No.09/446,952, filed Mar. 27, 2000 (now U.S. Pat. No. 6,387,369, issued May14, 2002), which is the national phase application of PCT ApplicationNo. PCT/US98/14520, filed Jul. 14, 1998, which claims priority of U.S.provisional application Ser. No. 60/052,910, filed Jul. 14, 1997; thecontents of each application are incorporated by reference in theirentireties.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[Not Applicable]

BACKGROUND OF THE INVENTION

The present technology relates to the replacement, repair, and/orregeneration of cardiac tissue and muscle.

On a yearly basis, it is estimated over 300,000 Americans will die fromcongestive heart failure. The ability to augment weakened cardiac musclewould be a major advance in the treatment of cardiomyopathy and heartfailure. Despite advances in the medical therapy of heart failure, themortality due to this disorder remains high, where most patients diewithin one to five years after diagnosis.

A common heart ailment in the aging population is improper heart valvefunction, particularly the aortic valve. Mechanical replacement valvesare widely used, but require the patient to continually take bloodthinners. Valves obtained from cadavers and xenographs (porcine) arealso frequently used to replace a patient's own tissue. Such valves arefreeze-dried or chemically cross-linked using, for example,glutaraldehyde to stabilize the collagen fibrils and decreaseantigenicity and proteolytic degradation. However, these valves remainacellular and often fail after several years due to mechanical strain orcalcification. A replacement valve derived from a biocompatible materialthat would allow ingrowth of the appropriate host cells and renewal oftissue over time would be preferred.

Adult bone marrow is an accessible and renewable source of adult stemcells that can be greatly expanded in culture. For example, mesenchymalstem cells (MSCs) are multipotential cells that have been identified andcultured from avian and mammalian species including mouse, rat, rabbit,dog and human (See Caplan, 1991; Caplan et al. 1993; and U.S. Pat. No.5,486,359). Isolation, purification and culture expansion of hMSCs isdescribed in detail therein.

BRIEF SUMMARY OF THE INVENTION

In accordance with the present technology, adult bone marrow-derivedstem cells are used to regenerate or repair striated cardiac muscle thathas been damaged through disease or degeneration. Adult bonemarrow-derived stem cells of the present technology have the capacity todifferentiate into at least one cell type of each of the mesodermal,ectodermal, and endodermal lineages. For example, the cells can beinduced to differentiate into cells of at least osteoblast, chondrocyte,adipocyte, fibroblast, marrow stroma, skeletal muscle, smooth muscle,cardiac muscle, endothelial, epithelial, hematopoietic, glial, neuronalor oligodendrocyte cell types, among others. Adult bone marrow-derivedstem cells of the present technology are capable of differentiating intoat least one cell type of at least one of the endodermal, ectodermal, ormesodermal embryonic lineages.

The present technology also provides a cell preparation comprising adultbone marrow derived stem cells in a dose effective to stimulateangiogenesis in the heart of a subject in need thereof. The stem cellscan be capable of differentiating into at least one cell type of each ofthe endodermal, ectodermal, and mesodermal embryonic lineages. Theeffective dose of the cell preparation can comprise about 0.02 to about150 milliliters of a pharmaceutically acceptable liquid medium. The stemcells can be at a concentration of about 10 to about 40 million cellsper milliliter. The stem cells can have an induced expression of atleast one cardiac specific marker, which can be, for example, myosinheavy chain, sarcoplasmic reticulum calcium ATPase, or connexin 43. Theinduced expression of the cardiac specific marker can result from (i)co-culturing the stem cells with cardiac cells, (ii) use of chemicalfusigens to create heterokaryons of the stem cells with cardiomyocytes,(iii) incubating the stem cells with extracts of mammalian hearts, (iv)treatment of the stem cells with growth factors, differentiating agents,or both, (v) mechanical or electrical stimulation of the stem cells, or(vi) mechanical or electrical coupling of the stem cells withcardiomyocytes. The subject can be, for example, a myocardial infarctionpatient, an ischemic heart transplant patient, a congestive heartfailure patient, or a coronary artery bypass graft patient. The cellpreparation can be capable of improving ventricular ejection fraction(e.g., left ventricular ejection fraction) in the subject.

The present technology further provides a method of improvingventricular ejection fraction in a patient comprising administering tothe patient bone marrow-derived stem cells in an amount effective toimprove ventricular ejection fraction. Again, the ventricular ejectionfraction can be left ventricular ejection fraction. The stem cells canbe autologous or allogeneic to the patient. The stem cells can beadministered in a pharmaceutically acceptable carrier, such as a liquidcarrier. The stem cells can be administered directly to the heart of thepatient, such as via a suitable catheter or during an open surgicalprocedure (e.g., coronary artery bypass graft surgery). The stem cellscan be administered systemically, such as intravenously orintraarterially. The stem cells can be mesenchymal stem cells.

The present technology also provides a method of treating a patienthaving myocardial infarction or congestive heart failure and havingreduced cardiac function comprising the step administering to thepatient bone marrow-derived stem cells in an amount effective to improvecardiac function. The stem cells can be autologous or allogeneic to thepatient. The stem cells can be administered in a pharmaceuticallyacceptable carrier, such as a liquid carrier. The stem cells can beadministered directly to the heart of the patient, such as via asuitable catheter or during an open surgical procedure (e.g., coronaryartery bypass graft surgery). The stem cells can be administeredsystemically, such as intravenously or intraarterially. The stem cellscan be mesenchymal stem cells. The method can further comprise assessingcardiac function at a time after administering the stem cells. Theassessment can comprise the step of measuring ventricular function, suchas by measuring ventricular ejection fraction, ventricular end diastolicpressure, or ventricular wall motion. The method can also comprisemeasuring left ventricular ejection fraction.

Additionally, the present technology provides a method of augmentingventricular function in a patient comprising administering to the heartof the patient a cell suspension, wherein the cell suspension comprisesbone marrow-derived stem cells and a pharmaceutically acceptable liquidmedium. The stem cells can be autologous or allogeneic to the patient.The suspension cells can be administered to the heart of the patient,such as via a suitable catheter or during an open surgical procedure(e.g., coronary artery bypass graft surgery). The stem cells can bemesenchymal stem cells. The patient can have, for example, a leftventricular ejection fraction of less than about 40%, or be sufferingfrom congestive heart failure.

In accordance with the present technology adult bone marrow-derived stemcells, such as mesenchymal stem cells (MSCs), are used to regenerate orrepair striated cardiac muscle that has been damaged through disease ordegeneration. In certain embodiments, MSCs differentiate into cardiacmuscle cells and integrate with the healthy tissue of the recipient toreplace the function of the dead or damaged cells, thereby repairingand/or regenerating the cardiac muscle as a whole. Cardiac muscle doesnot normally have reparative potential. Adult bone marrow-derived stemcells are used, for example, in cardiac muscle repair and/orregeneration for a number of principal indications: (i) ischemic hearttransplantations, (ii) therapy for congestive heart failure patients,(iii) prevention of further disease for patients undergoing coronaryartery bypass graft, (iv) conductive tissue regeneration, (v) vesselsmooth muscle regeneration, or (vi) valve regeneration. Adult bonemarrow-derived stem cells are also used to integrate with tissue of areplacement heart valve to be placed into a recipient. For example,MSCs, preferably autologous, repopulate the valve tissue, therebyenabling proper valve function.

Cardiac muscle therapy using adult bone marrow-derived stem cells isbased, for example, on the following sequence: harvest of stemcell-containing tissue, isolation/expansion of stem cells,administration by either implantation (including by catheterization)into the damaged heart (with or without a stabilizing matrix andbiochemical manipulation) or systemic injection, and in situ formationof myocardium or repair of damaged myocardium. This approach isdifferent from traditional tissue engineering, in which the tissues aregrown ex vivo and implanted in their final differentiated form.Biological, bioelectrical and/or biomechanical triggers from the hostenvironment may be sufficient, or under certain circumstances, may beaugmented as part of the therapeutic regimen to establish a fullyintegrated and functional repair or regenerated tissue.

Accordingly, one aspect of the present technology provides a method forproducing cardiomyocytes in an individual in need thereof whichcomprises administering to the individual a myocardium-producing amountof mesenchymal stem cells. The mesenchymal stem cells that are employedmay be a homogeneous composition or may be a mixed cell populationenriched in MSCs. Homogeneous human mesenchymal stem cell compositionsare obtained by culturing adherent marrow or periosteal cells; themesenchymal stem cells may be identified by specific cell surfacemarkers which are identified with unique monoclonal antibodies. A methodfor obtaining a cell population enriched in mesenchymal stem cells isdescribed, for example, in U.S. Pat. No. 5,486,359.

Adult stem cells can be obtained from a variety of sources, includingbone marrow. For example, human mesenchymal stem cells can be obtainedfrom bone marrow from a number of different sources, including plugs offemoral head cancellous bone pieces, patients with degenerative jointdisease during hip or knee replacement surgery, and aspirated marrowfrom normal donors or oncology patients who have marrow harvested forfuture bone marrow transplantation. Harvested marrow can be prepared forcell culture by a number of different mechanical isolation processesdepending upon the source of the harvested marrow (i.e., the presence ofbone chips, peripheral blood, etc.) that are well known in the art.Exemplary culture media and culture conditions are identified in, forexample, U.S. Pat. No. 5,486,359 and include media and conditions thatallow for expansion, growth, and isolation of mesenchymal stem cells,without differentiation.

Cell preparations having greater than about 95%, usually greater thanabout 98%, of adult human bone marrow-derived stem cells can be achievedusing techniques for isolation, purification, and culture expansion ofstem cells. For example, isolated, cultured mesenchymal stem cells cancomprise a single phenotypic population (about 95% or about 98%homogeneous) by flow cytometric analysis of expressed surface antigens.The desired cells in such composition can be identified, for example, byexpression of a cell surface marker (e.g., CD73 or CD105) specificallybound by an antibody produced from hybridoma cell line SH2, ATCCaccession number HB 10743, an antibody produced from hybridoma cell lineSH3, ATCC accession number HB 10744, or an antibody produced fromhybridoma cell line SH4, ATCC accession number HB 10745. Such antibodiesselectively bind bone marrow-derived mesenchymal stem cells and,therefore, can be used to identify, quantify, isolate, or purifymesenchymal stem cells from bone marrow samples.

The administration of the cells can be directed to the heart, by avariety of procedures. Localized administration is preferred. The adultbone-marrow derived stem cells can be from a spectrum of sourcesincluding, in order of preference: autologous, allogeneic, orxenogeneic. There are several embodiments to this aspect, including thefollowing.

In one embodiment of this aspect, the adult bone-marrow derived stemcells can be administered as a cell suspension in a pharmaceuticallyacceptable liquid medium for injection. Injection, in this embodiment,can be local, such as by administration directly into the damagedportion of the myocardium, or systemic, such as intravenously orintraarterially. Here, again, localized administration is preferred.

In another embodiment of this aspect, the adult bone-marrow derived stemcells, such as MSCs, are administered in a biocompatible medium whichis, or becomes in situ at the site of myocardial damage, a semi-solid orsolid matrix. For example, the matrix may be (i) an injectible liquidwhich “sets up” (or polymerizes) to a semi-solid gel at the site of thedamaged myocardium, such as collagen and its derivatives, polylacticacid or polyglycolic acid, or (ii) one or more layers of a flexible,solid matrix that is implanted in its final form, such as impregnatedfibrous matrices. The matrix can be, for example, Gelfoam (Upjohn,Kalamazoo, Mich.). The matrix holds the stem cells in place at the siteof injury, and thus serves the function of “scaffolding”. This, in turn,enhances the opportunity for the administered the adult bone-marrowderived stem cells to proliferate, differentiate and eventually becomefully developed cardiomyocytes. Although not wishing to be bound by anyparticular theory, it is believed that as a result of their localizationin the myocardial environment they then integrate with the recipient'ssurrounding myocardium. These events likewise occur in the above liquidinjectible embodiment, but this embodiment may be preferred where morerigorous therapy is indicated.

In another embodiment of this aspect, the adult bone marrow-derived stemcells are induced or genetically modified or engineered to expressproteins of importance for the differentiation and/or maintenance ofstriated muscle cells. Some examples include growth factors (TGF-β,IGF-1, FGF), myogenic factors (myoD, myogenin, Myf5, MRF), transcriptionfactors (GATA-4), cytokines (cardiotrophin-1), members of the neuregulinfamily (neuregulin 1, 2 and 3) and homeobox genes (Csx, tinman, NKxfamily). Also contemplated are genes that encode for factors thatstimulate angiogenesis and revascularization (e.g., vascular endothelialgrowth factor (VEGF)). Any of the known methods for introducing DNA aresuitable, however electroporation, retroviral vectors andadeno-associated virus (AAV) vectors are preferred.

Thus, in association with the embodiment of the above aspect usinggenetically engineered stem cells, the present technology also providesnew and unique genetically engineered adult bone marrow-derived stemcells and tissue compositions to treat the above indications. Thecompositions can include genetically modified cells and unmodified cellsin various proportions to regulate the amount of expressed exogenousmaterial in relationship to the total number of cells to be affected.

The present technology also relates to the potential of MSCs todifferentiate partially to the cardiomyocyte phenotype using in vitromethods. This technique can under certain circumstances optimizeconversion of MSCs to the cardiac lineage by predisposing them thereto.This also has the potential to shorten the time required for completedifferentiation once the cells have been administered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show cardiac muscle injected, using a fine needle, with invitro dye-labeled MSCs. The lipophilic dyes PKH26 (Sigma Chemical) orCM-Dil (Molecular Probes) were utilized to label MSCs prior to beingintroduced into animals. These dyes remain visible when the tissue siteis harvested approximately 1-2 months later. Such dyes do not interferewith the differentiation of MSCs in in vitro assays. FIG. 1A shows thelow magnification image of a rat heart which has been injected with dyelabeled cells and later, a T-incision was made at the site. FIGS. 1A and1B reveal the labeled MSCs in the ventricle wall viewed from the outersurface. FIG. 1C shows a cross-section of the ventricle wall and thatthe cells are present in the outer 1-2 mm of the 3 mm thick cardiacmuscle.

FIG. 2. Comparison of MSC engraftment when delivered to rats via directcardiac injection (Panel A) or tail vein (Panel B). Confocal images wereobtained in hearts harvested approximately 4 weeks post-implantation.

FIG. 3 shows images indicative of anterior wall motion in infarctedswine hearts that received no treatment and those that were treated withallogeneic MSCs.

FIG. 4 shows graphs of ejection fraction (upper panels) measured ininfarcted swine hearts that received no treatment and those that weretreated with MSCs, and graphs of global wall motion (lower panels) ininfarcted swine hearts that received no treatment, and those that weretreated with MSCs.

FIG. 5 is a graph of end diastolic pressure in infarcted swine heartsthat received no treatment and those that were treated with MSCs.

FIGS. 6A and 6B show sections of an infarcted region of a pig heart at 8weeks after being treated with DAPI-labeled mesenchymal stem cells. Bothfigures show the presence of blood vessels in the infarcted region. FIG.6A is a hematoxylin and eosin stained section, while FIG. 6B is afluorescent image showing the mesenchymal stem cells (dark, or blue) andof smooth muscle actin (light, or green), wherein the section wascontacted with an FITC-labeled monoclonal antibody against smooth muscleactin.

FIGS. 7A through 7E show sections of an infarcted pig heart at 12 weeksafter being treated with DAPI-labeled mesenchymal stem cells. Thefigures show the presence of blood vessels in the infarcted region.FIGS. 7A and 7D are hematoxylin and eosin stained sections. FIG. 7B is afluorescent image of DAPI-labeled mesenchymal stem cells. FIG. 7C is afluorescent image showing the presence of DAPI-labeled mesenchymal stemcells (dark, or blue) and of Factor VIII (light, or green), wherein thesection was contacted with an FITC labeled monoclonal antibody againstFactor VIII. FIG. 7E is a fluorescent image showing the presence ofDAPI-labeled mesenchymal stem cells (dark, or blue), and of vascularendothelial growth factor (VEGF), wherein the section was contacted withan FITC-labeled monoclonal antibody against VEGF.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

It is believed that he proper environmental stimuli convert MSCs intocardiac myocytes. Differentiation of mesenchymal stem cells to thecardiac lineage is controlled by factors present in the cardiacenvironment. Exposure of MSCs to a simulated cardiac environment directsthese cells to cardiac differentiation as detected by expression ofspecific cardiac muscle lineage markers. Local chemical, electrical andmechanical environmental influences alter pluripotent MSCs and convertthe cells grafted into the heart into the cardiac lineage.

Early in embryonic development following the epithelia-mesenchymetransition, the presumptive heart mesenchyme from the left and rightsides of the body migrate to the ventral midline. Here, interaction withother cell types induces continued cardiogenesis. In vitro conversion ofMSCs to cardiomyocytes is tested by co-culture or fusion with murineembryonic stem cells or cardiomyocytes, treatment of MSCs with cardiaccell lysates, incubation with specific soluble growth factors, orexposure of MSCs to mechanical stimuli and electrical stimulation.

A series of specific treatments applicable to MSCs to induce expressionof cardiac specific genes are disclosed herein. The conditions areeffective on rat, canine and human MSCs. Treatments of MSCs include (1)co-culturing MSCs with fetal, neonatal and adult rat cardiac cells, (2)use of chemical fusigens (e.g., polyethylene glycol or sendai virus) tocreate heterokaryons of MSCs with fetal, neonatal and adultcardiomyocytes, (3) incubating MSCs with extracts of mammalian hearts,including the extracellular matrix and related molecules found in hearttissue, (4) treatment of MSCs with growth factors and differentiatingagents, (5) mechanical and/or electrical stimulation of MSCs, and (6)mechanically and/or electrically coupling MSCs with cardiomyocytes. MSCsthat progress towards cardiomyocytes first express proteins found infetal cardiac tissue and then proceed to adult forms. Detection ofexpression of cardiomyocyte specific proteins is achieved usingantibodies to, for example, myosin heavy chain monoclonal antibody MF 20(MF20), sarcoplasmic reticulum calcium ATPase (SERCA1) (mAb 10D1) or gapjunctions using antibodies to connexin 43.

Cardiac injury promotes tissue responses which enhance myogenesis usingimplanted MSCs. Thus, MSCs are introduced to the infarct zone to reducethe degree of scar formation and to augment ventricular function. Newmuscle is thereby created within an infarcted myocardial segment. MSCsare directly infiltrated into the zone of infarcted tissue. Theintegration and subsequent differentiation of these cells ischaracterized, as described above. Timing of intervention is designed tomimic the clinical setting where patients with acute myocardialinfarction would first come to medical attention, receive first-linetherapy, followed by stabilization, and then intervention withmyocardial replacement therapy if necessary.

Of the four chambers of the heart, the left ventricle is primarilyresponsible for pumping blood under pressure through the body'scirculatory system. It has the thickest myocardial walls and is the mostfrequent site of myocardial injury resulting from congestive heartfailure. The degree of advance or severity of the congestive heartfailure ranges from those cases where heart transplantation is indicatedas soon as a suitable donor organ becomes available to those wherelittle or no permanent injury is observed and treatment is primarilyprophylactic.

The severity of resulting myocardial infarction as determined by thepercentage of muscle mass of the left ventricle that is involved canrange from about 5 to about 40 percent. This represents affected tissueareas, whether as one contiguous ischemia or the sum of smaller ischemiclesions, having horizontal affected areas from about 2 cm² to about 6cm² and a thickness of from 1-2 mm to 1-1.5 cm. The severity of theinfarction is significantly affected by which vessel(s) is involved andhow much time has passed before treatment intervention is begun.

The mesenchymal stem cells used in accordance with the presenttechnology are, in order of preference, autologous, allogeneic orxenogeneic, and the choice can largely depend on the urgency of the needfor treatment. A patient presenting an imminently life threateningcondition may be maintained on a heart/lung machine while sufficientnumbers of autologous MSCs are cultured or initial treatment can beprovided using other than autologous MSCs.

The MSC therapy of the present technology can be provided by severalroutes of administration, including the following. First, intracardiacmuscle injection, which avoids the need for an open surgical procedure,can be used where the MSCs are in an injectible liquid suspensionpreparation or where they are in a biocompatible medium which isinjectible in liquid form and becomes semi-solid at the site of damagedmyocardium. A conventional intracardiac syringe or a controllablearthroscopic delivery device can be used so long as the needle lumen orbore is of sufficient diameter (e.g., 30 gauge or larger) that shearforces will not damage the MSCs. The injectible liquid suspension MSCpreparations can also be administered intravenously, either bycontinuous drip or as a bolus. During open surgical procedures,involving direct physical access to the heart, all of the describedforms of MSC delivery preparations are available options.

As a representative example of a dose range is a volume of at leastabout 20 μl, preferably at least about 500 μl, of injectible suspensioncontaining about 10 to about 40×10⁶ MSCs/ml. The concentration of cellsper unit volume, whether the carrier medium is liquid or solid remainswithin substantially the same range. The amount of MSCs delivered willusually be greater when a solid, “patch” type application is made duringan open procedure, but follow-up therapy by injection will be asdescribed above. The frequency and duration of therapy will, however,vary depending on the degree (percentage) of tissue involvement, asalready described (e.g., about 5 to about 40% left ventricular mass).

In cases having about 5% to about 10% tissue involvement, it ispossible, for example, to treat with as little as a singleadministration of about one million MSCs in about 20 to about 50 μl ofinjection preparation. The injection medium can be any pharmaceuticallyacceptable isotonic liquid. Examples include phosphate buffered saline(PBS), culture media such as Dulbecco's Modified Eagle's Medium (DMEM;preferably serum-free), physiological saline or about 5% dextrose inwater (D5W).

In cases having more in a range of about 20% tissue involvement severitylevel, multiple injections of about 20 to about 50 μl (about 10 to about40×10⁶ MSCs/ml) are envisioned. Follow-up therapy may involve additionaldosings.

In very severe cases, for example in a range of about 40% tissueinvolvement severity level, multiple equivalent doses for a moreextended duration with long term (up to several months) maintenance doseaftercare may well be indicated and are envisaged in accordance with thepractice of the present technology.

When given intravenously, the mesenchymal stem cells may be administeredin at least about 20 μl, preferably at least about 500 μl, and up toabout 150 ml of a suspension containing about 10 to about 40×10⁶MSCs/ml. In one embodiment, from about 40 ml to about 150 ml of asuspension containing about 10 to about 40×10⁶ MSCs/ml is givenintravenously.

Applicants also have discovered that the mesenchymal stem cells maystimulate and/or promote angiogenesis in the heart and/or repair orregenerate blood vessels of the heart. Thus, in accordance with anotheraspect of the present technology, there is provided a method ofstimulating or promoting angiogenesis in the heart, and/or of repairingor regenerating blood vessels of the heart of an individual byadministering to the individual mesenchymal stem cells in an amounteffective to stimulate or promote angiogenesis, and/or repair orregenerate blood vessels of the heart. The mesenchymal stem cells may beadministered as a cell suspension in a pharmaceutically acceptableliquid medium such as described herein, or in a biocompatible mediumwhich is, or becomes in situ at the site of myocardial damage, asemi-solid or solid matrix, also as described herein.

The mesenchymal stem cells may be allogeneic, autologous, or xenogeneic,and may be administered in dosages such as those described herein.

When the mesenchymal stem cells are administered as a cell suspension ina pharmaceutically acceptable liquid medium for injection, they may beadministered locally, such as by administration directly into thedamaged portion of the heart by, for example, an endocardial catheter,or they may be administered systemically, such as by intravenous orintraarterial administration.

Adult bone marrow-derived stem cells, including mesenchymal stem cells,provide for the repair and/or regeneration of existing blood vessels ofthe heart, as well as promote angiogenesis (i.e., the formation of newblood vessels of the heart). Blood vessels which may be repaired orregenerated, as well as new blood vessels which may be formed, includearteries (including arterioles), and veins, as well as capillaries.

The present technology is further illustrated, but not limited, by thefollowing examples.

Example 1 Implantation of MSCs in Normal Cardiac Muscle

In using MSCs, it is desirable, for example, to maintain cell-cellcontact in vivo for the conversion of MSCs to the muscle lineage.Environmental signals identified above act in concert with mechanicaland electrical signaling in vivo to lead to cardiac differentiation.

Primary human MSCs (hMSCs) are introduced into athymic rat myocardialtissue by direct injection. The integration of implanted cells, theirsubsequent differentiation, formation of junctions with cardiac cells,and their long-term survival are characterized with light microscopy,histology, confocal immunofluorescence microscopy, electron microscopyand in situ hybridization.

Whether human MSCs are appropriately grafted into cardiac muscle ofathymic rats (strain HSD:RH-RNU/RNU), which lack the immune responsesnecessary to destroy many foreign cells, is also examined.

Rat MSCs are grafted into the heart muscles of rats. To analyze theinjected cells over several weeks and to minimize the possibility ofimmune system rejection, MSCs are harvested from Fisher 344 rats, thesame inbred strain (identical genotype) as the intended MSC recipients.

The MSCs can be marked in a variety of ways prior to their introductioninto the recipient. This makes it possible to trace the fate of the MSCsas they proliferate and differentiate in the weeks following the MSCimplant. Several methods are utilized to identify positively theinjected cells: membrane lipid dyes PKH26 or CM-Dil and genetic markingwith adeno-associated virus (AAV) or retroviruses, such as Moloneymurine leukemia virus expressing green fluorescent protein (GFP) orgalactosidase. PCR also is used to detect the Y chromosome marker ofmale cells implanted into female animals. The dye-labeled cells aredetected readily and offer the simplest method to directly follow theinjected cells. This method is reliable for times out to at least 4weeks. On the day of introduction to recipient animals, MSCs aretrypsinized and labeled with CM-Dil according to the recommendations ofthe manufacturer (Molecular Probes). Subconfluent monolayer cultures ofMSCs are incubated with 5 mM CM-Dil in serum-free medium for 20 minutes,trypsinized, washed twice in excess dye-free medium, and utilized forinjection.

Alternatively, MSCs are genetically marked prior to injections, such asby using AAV-GFP vector. This vector lacks a selectable marker, butmediates a high-level expression of the transduced genes in a variety ofpost-mitotic and stem cell types. Recombinant AAV-GFP is added to lowdensity monolayers of MSCs in low serum. Following a four hourincubation at 37° C., the supernatant is removed and replaced with freshmedia. At 96 hours after transduction, cells are assayed for greenfluorescent protein (GFP) activity. Typically 50% of the cells expressthe transduced gene. Unselected MSCs on a clonal line, isolated bylimiting dilution, are utilized for injection. Cells are collectedfollowing trypsin treatment, washed and used at high concentrations forinjection (10 to 100 million cells per ml).

To test whether the hMSCs became cardiomyocytes in the heartenvironment, the hearts of ten week old athymic rats were injected withdye labeled or GFP-labeled human MSCs. All procedures were performedunder strict sterile conditions. The animals were placed in a glass jarcontaining a methoxyflurane anesthesia soaked sponge. Under sterileconditions, a 20 mm anterior thoracotomy was performed, and followingvisualization of the left ventricle, about 10 μl of the cell suspension,containing about 10,000 to about 100,000 MSCs in serum-free medium wereinjected into the left ventricular apex using a 30 gauge needle. Theprocedure was performed rapidly with endotracheal intubation andmechanical ventilation assist. The incision was closed with sutures.Ventilation assist was normally unnecessary after a short periodfollowing chest closure. FIG. 1A shows the low magnification image of arat heart which was injected with dye labeled cells and later, aT-incision had been made at the site to reveal the injected cells in theventricle wall. FIG. 1A is a gross photo of the incised heart. FIGS. 1Band 1C reveal the labeled MSCs in the ventricle wall. FIG. 1C shows thatthe cells were present in the outer 1 to 2 mm of the 3 mm thick ratcardiac muscle.

When sacrificed, the heart is removed, examined by light microscopy forthe presence of vascular thrombi or emboli, paraffin-embedded, andsectioned. The histology of serial sections is examined to determine thefate of dye-stained cells. Sections then are tested forimmunohistochemical markers of cardiac muscle in the areas of theintroduced MSCs to ascertain whether donor MSCs have differentiated intocardiomyocytes in vivo. Implantation surgeries are carried out onanimals to be sacrificed at 1, 2, 4, and 6 weeks (4 animals at each timepoint) and the hearts which received implants are analyzedhistologically and immunologically.

For phenotypic characterization, the hearts are removed and processedfor histology by immunofluorescence microscopy. Differentiation of MSCsis determined by the immunofluorescence localization of sacomeric myosinheavy chain, SERCA1 and phospholamban. The sequence-specific antibody togap junction protein connexin 43, which is commercially available(Zymed, San Francisco, Calif.) and detects gap junctions in cardiactissue is also used.

MSCs are also implanted in biomatrix materials to determine if enhancedgrafting would be observed, such as Type I collagen. The MSCs are mixedrapidly with the matrix in a small volume and injected into theventricle wall. The biomatrices are used at concentrations of 0.1 mg/mlor greater. For example, the biomatrices may be used at concentrationsof 1 to 3 mg/ml containing 10 to 100 million cells/ml. The tissue isanalyzed at times of 1, 2, 4, and 6 weeks as described above.

Example 2 Regeneration of Heart Valves Using MSCs

Xenograft or homograft valves are made acellular by freeze-drying, whichleads to cellular death, or by enzymatic treatment followed by detergentextraction of cells and cell debris. This latter approach was taken byVesely and coworkers with porcine valves to be repopulated with dermalor aortic fibroblasts. Curtil, et al. 1997 used a freeze-dried porcinevalve and attempted repopulation of the valve with human fibroblasts andendothelial cells. These studies were preliminary and limited to shortterm studies in vitro.

The acellular valve to be populated by autologous hMSCs is incubatedwith culture expanded hMSCs in a tumbling vessel to ensure loading ofcells to all valve surfaces. The valve is then cultured with the hMSCsfor 1-2 weeks to allow the hMSCs to infiltrate and repopulate the valve.Within the culture vessel, the valve is then attached to a pump to allowthe actuation of the valve leaflets and simulate the pumping motionpresent in the body. The valve is maintained in the pumping mode for 1-2weeks to allow cellular remodeling associated with the stresses of thepumping action. Once sufficient cellular remodeling has occurred, thevalve is implanted into the body of the patient.

Another embodiment of this aspect of the present technology is to firstrepopulate the valve with hMSCs and to later incubate the valve tissueduring the pumping stage with autologous smooth muscle cells isolatedfrom a vascular graft which will line the lumen of the valve.

Example 3 MSC Engraftment in Rat MI Model: Direct Injection vs. SystemicDelivery

Myocardial Infarction was Produced in Fisher Rats as Follows:

Fisher rats were given a cocktail of Ketamine/Xylazine/Acepromazine (8.5mg/1.7 mg/0.3 mg I.P.) The depth of anesthesia was assessed using atoe-pinch and eye-blink reflexes. When a surgical plane of anesthesiawas achieved, endotracheal intubation was performed and the animalplaced on 1.0% Isoflorane. Positive-pressure breathing was providedthroughout the procedure by means of the Engler ADS 1000 small animalventilator. A left thoracotomy was performed and the pericardium opened.A 6-0 silk ligature snare was then placed around the left anteriordescending (LAD) coronary artery at a location distal to the firstdiagonal branch. A brief (30 sec) LAD test occlusion is performed toinsure that a modest region of ischemia is produced, involving a limitedregion of the anterior free wall and septum. Ischemia is confirmed bycharacteristic ECG changes, ventricular dyskinesis and regionalcyanosis. Myocardial infarction is then produced by occluding the LADfor a period of 45 minutes. At the completion of the 45 minute period,the snare is removed and reperfusion visually confirmed. The chest wasthen closed by approximating the ribs and all associated musculature.The Isoflurane is turned off, the animal removed from the ventilator andextubated.

Panel A of FIG. 2 shows engraftment of MSCs in the heart followingdirect injection into the heart. In these experiments, 2−4×10⁶allogeneic rat MSCs were implanted into the area of necrosis by directinjection.

Panel B of FIG. 2 shows that tail vein injection results in cardiacengraftment.

These animals received MSCs via the tail vein. Injection of theallogeneic cell suspension occurred when the animals had stabilized, anda normal cardiac rhythm had been reestablished; usually within 15minutes of reperfusion. At that time approximately 5×10⁶ MSCs in a 0.5milliliter suspension were injected slowly into the tail vein.

Example 4 Administration of MSCs Following MI Improves VentricularFunction

Swine are sedated with 1000 mg ketamine IM and brought into the lab.Intravenous access is established via an ear vein and the animalsanesthetized with nembutal. Swine then are intubated, ventilated with1.0-1.5% isoflurane, and prepped for surgery. ECG leads and rectaltemperature probes are placed and the animal is draped to create asterile field. A midline sternotomy is performed and the heart suspendedin a pericardial cradle. A tygon catheter is placed in the apex of theleft ventricle and sutured in place to measure ventricular pressurethroughout the cardiac cycle. The left anterior descending (LAD)coronary artery is dissected free just distal to the first diagonalbranch. A brief (30 sec) occlusion of the coronary artery is performedto identify the regions of ischemia (identified by the extent ofcyanosis). Four piezoelectric crystals then are placed within regionsdestined for infarction.

At the completion of the surgical instrumentation a 15 minutestabilization period is allowed prior to obtaining baseline recordings.Following these recordings, the LAD there is occluded for a period of 60minutes to produce myocardial infarction. Lidocaine (local anestheticand antiarrhythmic) is administered at this time to reduce the incidenceof ventricular fibrillation (2 mg/kg i.v. bolus plus 0.5 mg/min ivdrip). Recordings of left ventricular pressure and regional contractilefunction are again obtained at 10 and 50 minutes of ischemia. Extensivecyanosis within the ischemic bed was noticed following 50 minutes ofischemia.

At the completion of the 60 minute period of ischemia, the snare isreleased and reperfusion established. Care is taken to ensure thatperfusion is reestablished and that the isolated region of the LAD isnot in spasm. At this time the leads (sono leads and LV catheter) areexternalized, and the chest closed in layers. A chest tube is placed toreestablish a negative intrapleural pressure (tube is pulled 24 hrslater). The isoflurane is then turned off, and the animal is extubatedand allowed to recover.

One set of infarcted swine was treated with allogeneic mesenchymal stemcells and another set (control) did not receive such treatment. Theanimals were examined using echocardiography. In the mesenchymal stemcell treatment, a 10 ml MSC suspension was drawn up into several 3 ccsyringes using an 18 g needle. The needle was switched to a 30 g fordelivery. Implantation was accomplished in the open chest setting. Theneedle was advanced to the mid-wall level, and 0.5 mls of cells wereinjected. This same procedure was performed approximately 20 timesthroughout the damaged area. Care was taken to provide cells to theentire apical anterior wall, as well as the septum. At the completion ofthe implantation procedure, the chest was closed and the animal allowedto recover.

FIG. 3 contains “m-mode” images obtained in a control and an MSC treatedanimal. The image illustrates wall motion in a selected plane over time(moving left to right). The infarcted region of myocardium, consistingprimarily of anterior LV free wall, is the structure highlighted by thearrows. That segment of myocardium is essentially akinetic in thecontrol image, indicative of severe infarction/injury. While notquantifiable, there is improved anterior wall motion in the animaltreated with allogeneic MSCs.

Echocardiography was used to measure the ejection fraction, a measure ofglobal pump efficiency (a normal ejection fraction of 70% indicates that70% of the LV volume is pumped with each beat of the heart; EF<40% isindicative of heart failure). Ejection fraction data is shown in theupper panels of FIG. 4. Control animals demonstrated no significantimprovement in EF over the course of the study. In contrast, astatistically significant improvement in cardiac pump function wasobserved in MSC treated animals (right panel).

A similar graph was used to represent wall motion score index (lowerpanels of FIG. 4). In this analysis, 17 segments of the left ventriclewere examined for wall motion and scored on a scale of 1-5, with 1representing “normal” wall motion. These segments, comprising all areasof the ventricle, can then be averaged to gather an index of global wallmotion (i.e., global function). As with ejection fraction, nosignificant improvement in wall motion was observed in control animalsover time. MSC treated animals showed consistent and significantimprovements in wall motion scores over time (right panel).

Further evidence for improved cardiac function with MSC treatment isfound when end diastolic pressure (EDP) is examined. When cardiac pumpfunction is reduced following infarction, a pathologic increase in leftventricular EDP is observed. This increase in EDP is a clinicallyrelevant finding that is often predictive of the progression to heartfailure following infarction. As shown in FIG. 5, the EDP of controlswine rose from approximately 12 to 35 mmHg in the 6 months followinginfarction. The rise in EDP in animals treated with MSCs of the presenttechnology was significantly attenuated at all time points examinedpost-infarction.

Example 5 Administration of MSCs Prevents and/or Reverses CongestiveHeart Failure Following MI

Pathologic ventricular remodeling following myocardial infarction is amajor cause of heart failure. It was previously demonstrated thatautologous mesenchymal stem cells (MSC) augment local systolic wallthickening and prevent pathologic wall thinning. Based on in vitrostudies, it was hypothesized that MSCs may be immuno-privileged, andthat implantation of allogeneic MSCs could prevent pathologic remodelingand improve cardiac performance in a swine model of myocardialinfarction. Piezoelectric crystals and an LV catheter were implanted indomestic swine prior to a 60′ LAD occlusion to produce infarction.Following reperfusion, treated animals (n=7) were injected withallogeneic Dil-labeled MSCs (2×10⁸ cells in 9 ml) throughout the regionof infarction. Control (CON, n=6) received vehicle injections.Allogeneic donor MSCs were previously isolated from swine iliac crestbone marrow, expanded in culture, and cryopreserved until the time ofimplantation. Hemodynamic parameters and regional wall motion wereevaluated in conscious animals bi-weekly using trans-thoracicechocardiography and sonomicrometry. Animals were sacrificed at varioustime points (6-24 weeks) and tissue harvested for histologicalexamination.

It was observed that implantation of allogeneic MSCs was not associatedwith ectopic tissue formation, significant inflammatory response or anyadverse clinical event. Robust engraftment of allogeneic MSCs wasobserved in all treated animals. Furthermore, engrafted MSCs were foundto express numerous muscle specific proteins, and exhibitedmorphological changes consistent with myogenesis. A marked improvementin both ejection fraction (55±9.4% vs 32.5±12.5% in CON) and global wallmotion score (1.45±0.15 vs 2.1±0.2 in CON) was observed in treatedanimals at 10 weeks post-MSC implantation. Systolic wall thickening anddiastolic wall thickness were also augmented in MSC treated animals.Because no significant difference in infarct size or cardiac loading wasnoted between groups, improvements in cardiac function are likelyattributable to MSC implantation. In conclusion, this example suggeststhat implantation of allogeneic MSCs at reperfusion may be an effectivetherapeutic option to prevent or reverse the progression to heartfailure following infarction.

The above examples illustrate that mesenchymal stem cells augmentventricular function, as shown, for example by improved cardiac ejectionfraction and global wall motion.

Example 6 MSCs Repair and/or Regenerate Blood Vessels

A pig was subjected to a 60 minute LAD occlusion to produce infarctionas described in Example 5. Three days after the infarction, 200×10⁶diaminopropidium iodide (DAPI)-labeled allogeneic mesenchymal stem cellswere administered to the left venticular wall by endocardial catheter as20 separate injections of 10×10⁶ cells each in 0.5 ml physiologicalsaline. DAPI is a nuclear stain which emits a strong blue fluorescenceand aids in the identification of implanted cells.

Eight weeks after administration of the mesenchymal stem cells, the pigwas sacrificed, and the heart was harvested for histologicalexamination. Sections were subjected to hematoxylin and eosin staining,or to fluorescence imaging after being contacted with an FITC-labeledmonoclonal antibody against smooth muscle actin.

The hematoxylin and eosin image (FIG. 6A) clearly illustrates thepresence of blood vessels within a generalized region of myocardialnecrosis.

As shown in FIG. 6B, DAPI-labeled cells can be seen throughout thesection; however, a localization of implanted MSCs can be identifiedreadily. These MSCs surround, and are associated with, the bloodvessels.

As shown in FIG. 6B, the lighter, or green, fluorescence indicates thepresence of the FITC-labeled monoclonal antibody against smooth muscleactin, thus indicating the presence of a blood vessel. Also present inFIG. 6B are DAPI-labeled (blue) MSCs localized within such vessel, andwhich are associated intimately with the smooth muscle layer of thevessel. Thus, the MSCs are involved in the repair or regeneration ofblood vessels of the heart.

Example 7 Cardiac Engrafted MSCs Express Angiogenic Proteins

A pig was subjected to a 60 minute LAD occlusion to produce infarctionas described in Example 5. Three days after the infarction; the pig wasgiven 200×10⁶ diaminopropidium iodide (DAPI)-labeled allogeneicmesenchymal stem cells as 20 separate injections of 10×10⁶ cells in 0.5ml physiological saline into the left ventricular wall by endocardialcatheter as described in Example 6.

Twelve weeks after administration of the mesenchymal stem cells, the pigwas sacrificed, and the heart was harvested for histologicalexamination. Sections were subjected to hematoxylin and eosin staining,or to fluorescence imaging after being contacted with an FITC-labeledmonoclonal antibody against Factor VIII (Von Willebrand Factor) or anFITC-labeled monoclonal antibody against vascular endothelial growthfactor (VEGF).

As shown in the hematoxylin and eosin images of FIGS. 7A and 7D, thepresence of blood vessels within a region of generalized myocardialnecrosis is illustrated.

In the fluorescent images of FIGS. 7B, 7C and 7E, DAPI labeled cells canbe seen throughout the sections; however, localizations of MSCs can beidentified which surround and are associated intimately with the smoothmuscle layer of the blood vessels. Light, or green, fluorescenceindicates the presence of FITC-labeled monoclonal antibody againstFactor VIII (FIG. 7C) or against VEGF (FIG. 7E).

Thus, it has been shown that the implanted MSCs express Factor VIII andVEGF, which are indicative of angiogenesis. These proteins are notexpressed by cultured MSCs, but are expressed only after several weeksin the cardiac environment.

The disclosure of all patents and publications (including publishedpatent applications) are hereby incorporated by reference in theirentireties to the same effect as if each patent and publication wereindividually and specifically incorporated by reference.

It is to be understood, however, that the scope of the presenttechnology is not to be limited to the specific embodiments describedabove. The technology may be practiced other than as particularlydescribed and still be within the scope of the accompanying claims.

1. A method of improving left ventricular ejection fraction in a patientcomprising the step of: administering to the patient adult allogeneicbone marrow-derived stem cells in an amount effective to improveventricular ejection fraction.
 2. The method of claim 1, wherein thestem cells are administered intravenously or intraarterially.
 3. Themethod of claim 1, wherein the stem cells are administered in apharmaceutically acceptable liquid carrier.
 4. The method of claim 3,wherein the pharmaceutically acceptable carrier comprises an isotonicliquid.
 5. The method of claim 4, wherein the isotonic liquid comprisesphosphate buffer saline, physiological saline, culture medium, or 5%dextrose in water.
 6. The method of claim 5, wherein the isotonic liquidis physiological saline.
 7. The method of claim 1, wherein the adultallogeneic bone marrow-derived stem cells are administered in asuspension containing about 10×10⁶ to about 40×10⁶ adult allogeneic bonemarrow-derived stem cells per ml.
 8. A method of treating a patienthaving myocardial infarction or congestive heart failure and havingreduced cardiac function comprising the step of: administering to thepatient adult allogeneic bone marrow-derived stem cells in an amounteffective to improve cardiac function.
 9. The method of claim 8, whereinthe stem cells are administered intravenously or intraarterially. 10.The method of claim 8, further comprising the step of assessing cardiacfunction at a time after administering the stem cells, wherein theassessing comprises measuring at least one of left ventricular ejectionfraction, ventricular end diastolic pressure, or ventricular wallmotion.
 11. The method of claim 10, wherein the adult allogeneic bonemarrow-derived stem cells are administered in a pharmaceuticallyacceptable liquid arrier.
 12. The method of claim 11, wherein thepharmaceutically acceptable carrier comprises an isotonic liquid. 13.The method of claim 12, wherein the isotonic liquid comprises phosphatebuffer saline, physiological saline, culture medium, or 5% dextrose inwater.
 14. The method of claim 10, wherein the mesenchymal stem cellsare administered in a suspension containing about 10×10⁶ to about 40×10⁶mesenchymal stem cells per ml.
 15. A method of augmenting ventricularfunction in a patient comprising the step of: administering to the heartof the patient a cell suspension, wherein the cell suspension comprisesadult allogeneic bone marrow-derived stem cells and a pharmaceuticallyacceptable liquid medium.
 16. The method of claim 15, wherein thepatient has a left ventricular ejection fraction of less than about 40%.17. The method of claim 15, wherein the stem cells are mesenchymal stemcells.
 18. The method of claim 15, wherein the adult allogeneic bonemarrow-derived stem cells are administered in a pharmaceuticallyacceptable liquid carrier.
 19. The method of claim 18, wherein thepharmaceutically acceptable carrier comprises an isotonic liquid. 20.The method of claim 15, wherein the adult allogeneic bone marrow-derivedstem cells are administered in a suspension containing about 10×10⁶ toabout 40×10⁶ adult allogeneic bone marrow-derived stem cells per ml.